1. Field of the Invention
The present invention relates generally to the field of the transport of serum proteins and antibodies mediated by the Fc receptor, FcRn, and further to the effect on serum half life of agents that interact with the Fc receptor in a pH dependent way.
2. Description of Related Art
IgGs constitute the most prevalent immunoglobin class in the serum of man and other mammals and are maintained at remarkably constant levels. Recent studies indicate that the major histocompatibility complex (MHC)-class I related receptor, FcRn, is involved in the homeostasis of serum IgGs (Ghetie et al., 1996; Junghans and Anderson, 1996; Israel et al., 1996). This receptor most likely acts as a salvage receptor, and this would be consistent with its known ability to transcytose IgGs in intact form across the neonatal gut (Wallace and Rees, 1980; Rodewald and Kraehenbuhl, 1984) and yolk sac (Roberts et al., 1990; Israel et al., 1995) or placenta (Kristoffersen and Matre, 1996; Simister et al., 1996; Leach et al., 1996). The interaction site of FcRn on mouse IgG1(mIgG1) has been mapped using site-directed mutagenesis of recombinant Fc-hinge fragments, followed by analysis of these fragments both in vivo and in vitro(Kim et al., 1994b; Medesan et al., 1996; 1997). From these studies, I253 (EU numbering (Edelman et al., 1969)), H310, H435 and to a lesser extent, H436 play a central role in this interaction. These amino acids are located at the CH2-CH3 domain interface (Deisenhofer, 1981), and the mapping of the functional site to these residues is consistent with the X-ray crystallographic structure of rat FcRn complexed with rat Fc (Burmeister et al., 1994b).
The FcRn interaction site encompasses three spatially close loops comprised of sequences that are distal in the primary amino acid sequence. The central role of Fc histidines in building this site accounts for the marked pH dependence (binding at pH 6.0, release at pH 7.4) of the Fc-FcRn interaction (Rodewald and Kraehenbuhl, 1984; Raghavan et al., 1995; Popov et al., 1996), as the pKa of one of the imidazole protons lies in this pH range. I253, H310, H435 and to a lesser degree, H436, are highly conserved in IgGs of both human and rodent IgGs (Kabat et al., 1991). This, taken together with the isolation of a human homolog of FcRn (Story et al., 1994), indicate that the molecular mechanisms involved in IgG homeostasis are common to both mouse and man and this has implications for the modulation of the pharmacokinetics of IgGs for use in therapy.
To date, in studies to identify the FcRn interaction site on Fc, mutations of Fc-hinge fragments have been made that reduce the serum half lives of the corresponding Fc-hinge fragments (Medesan et al., 1997; Kim et al., 1994a). The correlation between serum half life and binding affinity for FcRn is excellent for these mutated Fc-hinge fragments (Kim et al., 1994b; Popov et al., 1996), suggesting that if the affinity of the FcRn-Fc interaction could be increased, whilst still retaining pH dependence, this would result in an Fc fragment with prolonged serum persistence. Production of such a fragment would be a significant advance in the engineering of a new generation of therapeutic IgGs with improved pharmacokinetics such as increased persistence in the circulation. But to date, no such fragments have been produced.
Immunoglobulin Fc domains are also of great interest for purposes of studying the mechanisms of antibody stabilization, catabolism and antibody interactions with further molecules of the immune system. These include, depending on the class of antibody, interactions with complement, and binding to specific receptors on other cells, including macrophages, neutrophils and mast cells. More detailed knowledge of the biology of Fe regions is important in understanding various molecular processes of the immune system, such as phagocytosis, antibody-dependent cell-mediated cytotoxicity and allergic reactions.
The production of a longer-lived Fc fragment that has increased binding to FcRn would be attractive, since such a fragment could be used to tag therapeutic reagents. Chimeric proteins produced in this manner would have the advantage of high in vivo stability which would allow fewer doses of the agent to be used in therapy and possibly even allow lower doses of the agent to be used through its increased persistence in the bloodstream. Unfortunately,methodology for generating proteins, such as antibody fragments, with increased serum persistence has not yet been developed.
The present invention seeks to overcome deficiencies in the art by providing functional proteins, antibodies or other agents that have an increased serum half-life through the interaction with Fc receptor (FcRn). These functional agents include any molecule that binds to FcRn in a pH dependent way such that binding affinity is strong at about pH 6 to about pH 6.5 relative to binding at pH 7.4. Physiologically, this allows the agent to be salvaged by FcRn at lower pH and released into the essentially neutral pH environment of the serum. The present disclosure includes protein and peptide compositions having altered serum half-lives relative to IgG, methods of making such proteins or peptides, either starting with a known sequence or by screening random sequences, and methods of screening unknown candidate agents for pH dependent FcRn binding. In addition, disclosed herein are methods of making an agent with altered serum half-life by conjugating or otherwise binding of that agent to a moiety identified as having an increased serum half-life through its interaction with FcRn. Such agents would include, but are not limited to antibodies, fragments of antibodies, hormones, receptor ligands, immunotoxins, therapeutic drugs of any kind, T-cell receptor binding antigens and any other agent that may be bound to the increased serum half life moieties of the present invention.
Also disclosed are methods of increasing the FcRn binding affinity of an FcRn binding protein or peptide so that the protein or peptide will have an increased serum half-life. These methods include identifying amino acids that directly interact with FcRn. These amino acids may be identified by their being highly conserved over a range of species, or by any other method. Other methods would include, for example, mutation or blocking of the amino acid and screening for reduced binding to FcRn, or by a study of three dimensional structure of the interaction, or by other methods known in the art. When those residues are identified that directly interact, then secondary amino acids are identified whose side chains are in the spatial vicinity of the direct interaction. In the case of antibodies, these secondary amino acids often occur in loops so that they are exposed to the solvent. In this way, mutation of these amino acids is not expected to disrupt the native protein structure. These identified secondary amino acids are then randomly mutated and the mutated proteins or peptides are then screened for increased binding affinity for FcRn at about pH 6 relative to the non-mutated protein or peptide. This method is applicable to any protein or peptide that binds FcRn in a pH dependent way and all such proteins or peptides would be encompassed by the present claimed invention. It is also understood that random mutation, in and of itself, does not constitute the invention, and that the secondary amino acids may be specifically mutated or modified or derivatized in any way known in the art and then screened for the effect on FcRn binding.
In certain broad aspects, the invention encompasses the design and production of recombinant antibody or antibody Fc-hinge domains engineered to have increased in vivo, or serum half lives. The Fc-hinge domain mutants with increased serum half lives of the present invention are generally defined as mutants in which one or more of the natural residues at the CH2-CH3 domain interface of the Fc-hinge fragment have been exchanged for alternate amino acids. Such Fc-hinge domain mutants may also be functionally defined as mutants which exhibit impaired SpA (Staphylococcal protein A) binding. In preferred embodiments, the increased half-life Fc-hinge mutants will have changes in certain amino acids between about residue 252 and about residue 436, which have been discovered to form, or be in close proximity to, the xe2x80x98catabolic control sitexe2x80x99.
In a further embodiment, the invention encompasses the isolation of peptides or agents that bind to FcRn with an affinity that may not necessarily be greater than that of the IgG:FcRn interaction yet the peptides or agents still have a measurably longer half life than a similar peptides or agents that do not bind to FcRn in a pH dependent manner as described herein. It is envisioned that such peptides or agents are useful as a stabilization xe2x80x9ctagxe2x80x9d for a therapeutic agent or protein.
More particularly, the present invention concerns mutant Ig domains and antibodies containing domains in which one or more of the following amino acids have been exchanged for other residues: threonine (thr) at position 252, threonine at position 254, threonine at position 256 SEQ ID NO:37 (position 256 corresponds to residue 21 in SEQ ID NO: 39) (wherein the amino acids are numbered according to Kabat et al., (1991)). To increase the half life of an Fc-hinge domain, or intact antibody, any of the above residues may be substituted for any other amino acid residue and then variants that have higher affinity for FcRn may be selected using bacteriophage display, for example, or by any other method known to those of skill in the art. Substitution can advantageously be achieved by any of the molecular biological techniques known to those of skill in the art, as exemplified herein below, or even by chemical modification.
Certain increased half-life antibodies or domains will be those which include one or more of the following substitutions on the Kabat numbering system, or their equivalents on different numbering systems: threonine (thr) 252 to leucine (leu) 252, threonine 254 to serine (ser) 254, threonine 256 to phenylalanine (phe) 256. An example as disclosed herein is the triple mutant termed LSF which contains the three mutations: threonine 252 to leucine 252, threonine 254 to serine 254, threonine 256 to phenylalanine 256.
The production of Fc-hinge domains with longer in vivo half lives is an advantageous development in that it further delineates the site for the control of IgG1 catabolism to a specific region of the Fc-hinge fragment, and in practical terms, it has several important applications. It allows the design and construction of antibody molecules, domains, or fragments, such as bivalent Fab fragments, with longer half lives. These would be generally useful in that the slower biological clearance times would result in fewer administrations of any antibody or vaccine such that fewer xe2x80x9cboosterxe2x80x9d vaccinations may be required. Furthermore, these molecules with longer half lives can be used to tag other therapeutic molecules, such as vaccine molecules. The catabolic site delineated in this invention is distinct from the ADCC and complement fixing sites. This is important as antibodies may be produced which are completely functional and which have longer half lives. Other important uses include, for example, antibody-based systemic drug delivery, the creation of immunotoxins with longer lives or even antibody-based immunotherapy for chronic illnesses or conditions such as hay fever or other allergic reactions, or treatment of T-cell mediated autoimmune disorders by anti-T-cell receptor antibodies or T-cell antigens.
The Fc-hinge domain mutants may also be employed in embodiments other than those involving clinical administration, for example, in the isolation of receptors involved in IgG catabolism. To this end, one may use screening assays or differential screening assays in which the mutants would exhibit binding or increased binding to a potential catabolic receptor.
The discoveries disclosed herein concerning antibody catabolism are also envisioned to be useful to increase the in vivo half life of virtually any recombinant protein, and particularly a recombinant antibody, which one desires to administer to a human or animal. An antibody or recombinant protein that was found to be cleared from the body more quickly than ideally desired could be engineered at the residues identified herein, or in the vicinity of amino acids that are discovered to directly interact with FcRn, such that its in vivo half life was increased.
In certain other embodiments, the present invention contemplates the creation of recombinant molecules, particularly antibody constructs, including vaccines and immunotoxins, with increased in vivo half lives. Longevity of recombinant molecules is often needed, and several protocols would benefit from the design of a molecule which would be more slowly removed from circulation after exerting its designed action. This may include, for example, antibodies administered for the purpose of scavenging pathogens, toxins or substances causing biological imbalances and thereby preventing them from harming the body; and antibodies designed to provide long-term, systemic delivery of immunotherapeutic drugs and vaccines.
To generate a domain, antibody or antibody construct with a longer half-life, one would modify the natural residues at the CH2-CH3 domain interface of the Fc-hinge which either form the xe2x80x9ccatabolic control sitexe2x80x9d or are in close proximity to it. Several such catabolism controlling mutations are described herein which may be straightforwardly engineered into an antibody molecule or antibody conjugate. These include, substituting another residue for threonine 252, threonine 254, threonine 256, methionine 309, glutamine 311 and/or asparagine 315 SEQ ID NO:37 (position 256 corresponds to reduce 80 in SEQ 10 No:39) (Kabat et al., 1991). The present invention also provides an advantageous method for determining other residues important for catabolism control.
The proteins or peptides of the present invention may be expressed from recombinant plasmids or expression vectors adapted for expression of immunoglobulin-like domains, such as antibody domains, or other proteins or peptides in recombinant host cells. Recombinant plasmids thus may comprise a DNA segment coding for one or more immunoglobulin-like domains. Accordingly, any one or more of a wide variety of immunoglobulin-like domains or other protein or peptide may be incorporated into a recombinant vector and expressed in a host cell in accordance herewith. These include, but are not limited to, variable or constant domains from IgG, IgM, IgA, IgD, IgE, T cell receptors, MHC class I or MHC class II, and also, CD2, CD4, CD8, CD3 polypeptides, Thy-1 and domains from the PDGF receptor, N-CAM or Ng-CAM.
In certain embodiments, the present invention concerns the expression and production of antibody constant domains. The production of antibody Fc-hinge, Fc, CH2-hinge or CH3 domains is preferred, with Fc-hinge or Fc domains being particularly preferred due to their longer in vivo half lives. In other instances, the production of Fc-hinge domains (or antibodies incorporating such domains) with mutations at thr 252, thr 254 or thr 256 is preferred as these have specifically longer half lives. Such mutants are exemplified by thr 252 to leu 252, thr 254 to ser 254 and thr 256 to phe 256.
Various segments or subfragments of any of the above domains, as well as other variable or constant domains, may also be employed in accordance herewith. These domains include, for example, the immunoglobulin domains CH 1. Variations of immunoglobulin domains other than those specifically described above also fall within the scope of the invention. Such variations may arise from naturally-occurring or genetically engineered mutations, such as point mutations, deletions and other alterations affecting one or more amino acids or the addition of amino acids at the N or C termini.
Furthermore, while the invention has been illustrated with murine FcRn and immunoglobulin fragments, similar strategies are applicable to immunoglobulin-like domains or other proteins or peptides from a variety of other species, including mammals such as rat, and more particularly, human immunoglobulin-like molecules. In light of the structural similarity of the immunoglobulin-like domains, and the conservation of the immunoglobulin superfamily throughout evolution, it is contemplated that the techniques of the present invention will be directly applicable to the expression and recombinant production of an immunoglobulin-like domain from any given species.
Other DNA segments may also be included linked to the immunoglobulin-like domains described. For example, one or more recombinant antibody variable domains of varying specificities may be linked to one or more antibody constant domains, immunoglobulin constant domains, or even other proteins, such as bacteriophage coat protein genes, hormones or antigens, including T-cell receptor antigens. The antibody constant domains of the present invention may also be combined with another immunoglobulin domain, or indeed, with any other protein. The immunoglobulin constant domains may be variously expressed as a single domain, such as a CH3 domain; or in combination with one, two, three or more domains, such as, for example, as a CH2-hinge domain, an Fc domain, or an entire Fc-hinge domain. In particular embodiments, discussed in more detail below, Fc or Fc-hinge domains may be linked to any protein to produce a recombinant fusion with enhanced biological stability, or certain mutants may be employed to create antibodies or fusion proteins with increased half lives.
Once expressed, any of the products herein could be radiolabeled or fluorescently labeled, or attached to solid supports, including sepharose or magnetic beads or synthetic bilayers such as liposomes. The products could also be linked to carrier proteins such as bovine serum albumin. The Fc constant domains, or constant domains in combination with other proteins, could also be linked synthetically to co-receptors such as the extracellular domains of CD4 or CD8.
Recombinant, or cloning, vectors are included in one aspect of the present invention. Such vectors and DNA constructs will be useful not only for directing protein expression, but also as for use as templates for in vitro mutagenesis. Vectors will generally include a leader sequence, preferably pelB (Better et al., 1988), although other leader sequences may be used, for example, alkaline phosphatase (phoA) or ompA. In a preferred embodiment, the pelB leader segment is modified with a unique restriction site, such as NcoI, allowing insertion of antibody variable domain genes. Introduction of such restriction sites is a convenient means of cloning in a DNA segment in the same reading frame as the leader sequence.
Modification of the leader sequence DNA may be achieved by altering one or more nucleotides employing site-directed mutagenesis. In general, the technique of site specific mutagenesis is well known in the art as exemplified by publications (Carter et al., 1985; Sambrook et al., 1989). As will be appreciated, the technique typically employs a phagemid vector which exists in both a single stranded and double stranded form. Alternatively, mutants may be generated by using the PCR(trademark). Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage (Messing et al., 1981) or pUC 119. These vectors are readily commercially available and their use is generally well known to those skilled in the art. Alternatively, methods of site-directed mutagenesis employing double stranded plasmids or phagemids and the like are also well known in the art and may also be used in the practice of the present invention.
Site directed mutagenesis in accordance herewith is performed by first obtaining a single stranded vector which includes within its sequence the DNA sequence encoding a leader sequence, pelB being used herewith. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example by the method of Narang et al., (1980). The primer is annealed with the single stranded vector and subjected to DNA polymerizing enzymes such as the E. coli polymerase I Klenow fragment. In order to complete the synthesis of the mutation bearing strand, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. The heteroduplex may be transformed into a bacterial cell, with E. coli. being preferred. Clones are screened using colony hybridization and radiolabeled mutagenic oligonucleotides to identify colonies which contain the mutated plasmid DNA (Carter et al., 1985). PCR(trademark) directed mutagenesis, using double-stranded DNA templates, is particularly suitable for generating increased half life mutants. PCR(trademark) mutagenesis typically involves the use of a primer encoding one or more alternate or random amino acid in one or more amplification reactions.
Constructs may also include a xe2x80x9ctagxe2x80x9d useful for isolation and purification of the expressed polypeptide product. Tags are relatively short DNA segments fused in-frame with a sequence encoding a desired polypeptide, such as polyhistidine, which have the function of facilitating detection, isolation and purification. For example, affinity peptides may be encoded by the segments, allowing isolation by selective binding to specific antibodies or affinity resins. Any of a number of tags may be used, including the c-myc tag, (his)6 tag, decapeptide tag (Huse et al., 1989), Flag(trademark) (Immunex) tags and so forth. A number of the tags are also useful for the detection of expressed protein using Western blotting (Ward et al., 1989; Towbin et al., 1979).
(His)6 tags, for example, are preferable for purifying secreted polypeptide products on affinity metal chromatography columns based on metals such as Ni2+. The (his)6 peptide chelates Ni2+ ions with high affinity. Polypeptide products containing these residues at the N or C termini bind to the affinity columns, allowing polypeptide impurities and other contaminants to be washed away as part of the purification process. Polypeptide products can then be eluted from the column with high efficiency using, for example, 250 mM imidazole.
Peptide tags, or linkers, may also be incorporated into the immunoglobin product. For single chain Fv or T cell receptor (TCR) fragments, preferred linker peptides include a 15-mer, for example, (gly4ser)3, or other linkers, such as those described in Filpula and Whitlow (1991).
As mentioned above, recombinant vectors of the present invention may also include DNA segments encoding various other proteins. In particular, it is envisioned that recombinant vectors encoding antibody Fc-hinge or Fc domains may also include DNA segments encoding other proteins, or fragments thereof, particularly where one wishes to produce the protein in a form that has a longer serum half life. It is envisioned that the serum stability of proteins or peptides intended for administration to animals or humans may be increased in this manner. Examples of such proteins or peptides include, for example, interleukin-2, interleukin-4, xcex3-interferon, insulin, T cell epitopes and the like, and even TCR Va Vxcex2. A variety of synthetic drugs could, likewise, be stabilized in this manner.
DNA segments encoding such proteins may be operatively incorporated into a recombinant vector, in frame with the Fc-based domain, whether upstream or downstream, in a position so as to render the vector capable of expressing a protein:Fc domain fusion protein (or a protein: Fc-hinge domain fusion protein). Techniques for the manipulation of DNA segments in this manner, for example, by genetic engineering using restriction endonucleases, will be known to those of skill in the art in light of both the present disclosure and references such as Sambrook et al. (1989).
The invention has been illustrated with prokaryotic host cells, but this is not meant to be a limitation. The prokaryotic specific promoter and leader sequences described herein may be easily replaced with eukaryotic counterparts. It is recognized that transformation of host cells with DNA segments encoding any of a number of immunoglobulin-like domains will provide a convenient means of producing fully functional proteins, such as for example, functional IgGs. Both cDNA and genomic sequences are suitable for eukaryotic expression, as the host cell will, of course, process the genomic transcripts to yield functional mRNA for translation into protein. Increased half life mutant domains and antibodies may be produced in glycosylated form in eukaryotic systems which fix complement, and mediate ADCC.
It is similarly believed that almost any eukaryotic expression system may be utilized for the expression of proteins and peptides of the present invention, e.g., baculovirus-based, COS cell-based, myeloma cell-based systems could be employed. Plasmid vectors would incorporate an origin of replication and an efficient eukaryotic promoter, as exemplified by the eukaryotic vectors of the pCMV series, such as pCMV5.
For expression in this manner, one would position the coding sequences adjacent to and under the control of the promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one positions the 5xe2x80x2 end of the translation initiation site of the translation reading frame of the protein between about 1 and about 50 nucleotides xe2x80x9cdownstreamxe2x80x9d of (i.e., 3xe2x80x2 of) the chosen promoter.
Where eukaryotic expression is contemplated, one will also typically desire to incorporate into the transcriptional unit, an appropriate polyadenylation site (e.g., 5xe2x80x2-AATAAA-3xe2x80x2) if one was not contained within the original cloned segment. Typically, the poly A addition site is placed about 30 to 2000 nucleotides xe2x80x9cdownstreamxe2x80x9d of the termination site of the protein at a position prior to transcription termination.
As used herein the term xe2x80x9cengineeredxe2x80x9d or xe2x80x9crecombinantxe2x80x9d cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding an immunoglobulin-like domain, has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinant gene that is introduced by transfection or transformation techniques. Engineered cells are thus cells having a gene or genes introduced through the hand of man.
Suitable host cells useful in the practice of the invention include gram-negative organisms and might include Serratia marcescens, Salmonella typhimurium and similar species. A particularly preferred host cell is Escherichia coli and the several variants of E. coli that are readily available and well known to those of skill in the art.
A particular aspect of the invention is a method for the production of immunoglobulin-like domains, such as, native or mutant antibody constant domains, or subfragments or fusion proteins thereof. To produce such domains or modified domains, a gram-negative microorganism host cell is first transformed with any of the disclosed recombinant vectors, and then cultured in an appropriate bacterial culture medium under conditions to allow expression of the immunoglobulin-like domain(s), which may be subsequently isolated.
Culturing typically comprises growing and induction. Growing is conveniently performed in such media as Luria broth plus 1% glucose, 4xc3x97TY (double strength 2xc3x97TY) plus 1% glucose, minimal media plus casamino acids and 5% w/v glycerol with temperatures in the range of 20xc2x0 C. to about 37xc2x0 C., preferably between 25-30xc2x0 C. In preferred embodiments, the media will contain a selection agent, such as ampicillin at a concentration of 0.1 mg/ml to select bacterial cells which contain the expression plasmid. Naturally, one will choose a particular selection agent in conjunction with the plasmid construct originally employed, as is known to those of skill in the art.
Induction of expression is typically performed at a point after growth has been initiated, usually after 12-16 hours at 30xc2x0 C. This length of time results in the cells being in the early stationary phase at the induction stage. If the growth media contains glucose, the cells are pelleted and washed prior to addition of an inducer, such as isopropylthiogalactopyranoside (IPTG) at a concentration of 0.1-1 mM, since glucose inhibits induction of expression. Again, a variety of other inducers may be employed, according to the vector construct originally used, as is known in the art. Cells may be grown for shorter periods prior to induction, for example for 6-10 hours, or to the mid-exponential stage of growth. Cells are induced for 5-28 hours. Five to six hours of induction is a preferred induction time if the protein is to be isolated from the periplasm, since longer induction times result in the protein leaking into the culture supernatant. However, it may be desirable to isolate product from the external medium, in which case one would prefer using longer induction times. Temperatures in the range of 20xc2x0 C. to 37xc2x0 C. may be used as growth and induction temperatures, with 25xc2x0 C. being a preferred induction temperature.
Isolating polypeptide products produced by the microbial host cell and located in the periplasmic space typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis, but preferably by osmotic shock. Once cells are disrupted, cells or cell debris may be conveniently removed by centrifugation or filtration, for example. The proteins may be further purified, for example, by affinity metallic resin chromatography when appropriate peptide tags are attached to the polypeptide products.
Alternatively, if the induction period is longer than 8 hours (at 25xc2x0 C., for example), so that the protein leaks into the culture supernatant, cells may be removed from the culture by centrifugation and the culture supernatant filtered and concentrated (for example, 10-20 fold). Concentrated supernatant is then dialyzed against phosphate buffered saline and separation achieved by column chromatography, such as affinity or adsorption chromatography. An example is separation through Ni2+-NTA-agarose to separate appropriately tagged proteins such as those carrying a (his)6 tag. When these tags are used in the construction of an expression vector, histidine tags are particularly preferred as they facilitate isolation and purification on metallic resins such as Ni+2-NTA agarose.
As used herein, the term xe2x80x9cbiologically stable proteinxe2x80x9d is intended to refer to a protein which has been modified resulting in increased serum half life with respect to the original protein. This term encompasses both known recombinant proteins and also proteins for which the recombinant form has not yet been reported. As such, increased biological stability may be measured with respect to the known or original recombinant protein, or with respect to the native protein. Biological stability may be measured by a variety of in vitro or in vivo means, for example, by using a radiolabeled protein and measuring levels of serum radioactivity as a function of time, or by assaying the levels of intact antibody (of known specificity) present in the serum using ELISA as a function of time, with a particularly preferred measure of increased biological stability being evidenced by increased serum half life and decreased clearance rates.
To produce a biologically stable recombinant protein in which the protein in question is linked to an antibody Fc-hinge domain or an antibody Fc domain, in accordance herewith, one may first prepare a recombinant vector capable of expressing a protein: Fc-hinge or protein:Fc domain fusion protein in a gram-negative host, as described hereinabove. One would then insert the recombinant vector into a gram-negative bacterium and culture the bacterium under conditions effective to allow the expression of the fusion protein. Following this, one may then proceed to isolate the fusion protein so produced, for example, using the methods of the present invention.
The above method is proposed for use in the generation of a series of therapeutic compounds with improved biological stability. Such compounds include, for example, interleukin-2, insulin, interleukin-4 and interferon gamma, or even T cell receptor Va Vxcex2. The recombinant Fc domains of this invention are also contemplated to be of use in stabilizing a wide range of drugs, which would likely alleviate the need for their repeated administration. However, the present methods are not limited solely to the production of proteins for human administration, and may be employed to produce large quantities of any protein with increased stability, such as may be used, for example, in immunization protocols, in animal treatment by veterinarians, or in rodent in vivo therapy models.
A mutant Fc-hinge domain has been generated in the present invention and is herein shown to have a dramatically increased in vivo half life in comparison to native domains. The present invention therefore further encompasses methods by which to produce antibodies or proteins with extended biological half lives. These methods include, firstly, coupling a protein or an antibody variable domain to an increased half life mutant domain of the present invention, as described above. To produce such antibodies or proteins one would prepare a recombinant vector capable of expressing the desired fusion or mutated protein, insert the vector into a gram-negative bacterium, culture it to allow expression and isolate the antibody or protein so produced. These techniques are applicable to any antibody or protein which one desires to have a longer biological half life, including antibodies and immunotoxins.
Another method of the invention, particularly suited to producing antibodies with increased serum half lives, is to simply modify a given antibody at one or more of the residues disclosed herein either at, or in proximity to, the catabolic control site. This may be achieved chemically, or by random or site-directed mutagenesis and recombinant production using any known production method. A preferred method is to replace the indicated residues with all of the remaining 19 residues and then select (using phage display if more than one residue is mutated simultaneously) mutants that have higher affinity for FcRn. The selected mutants should also bind to FcRn in a pH dependent manner as described herein, the pH can be controlled during the selection steps. This selection method also is applicable to random peptide libraries or or any other randomly mutated protein. Antibodies engineered in this manner may be single antibodies, domains, Fab fragments, or antibody conjugates such as immunotoxins and antibodies used for therapeutic regimens.
Also contemplated within the scope of the invention are recombinant immunoglobulin-like domain products, such as variable or constant antibody domains; antibodies, antibody constructs, antibody domains or immunotoxins with extended half lives; or domains from MHC molecules or cell signalling molecules such as CD2, CD4, CD8, CD3, N-CAM or Ng-CAM, or PDGF receptor domains, or fragments thereof. In preferred embodiments, these will include antibody constant domain products, such as Fc-hinge, Fc, CH2-hinge and CH3 domains; and antibody Fc-hinge domains engineered to have longer in vivo half lives, such as, for example, the LSF mutant. It will be appreciated that modification and changes may be made in the composition of these domains, for example by altering the underlying DNA, and still obtain a molecule having like or otherwise desirable characteristics. As such, biological functional equivalents of these immunoglobulin-like domains and mutants such as peptides and other randomly mutated proteins that bind to FcRn are also included within the scope of the present invention.
In general, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or receptor sites. Since it is the interactive capacity and nature of a protein that defines that protein""s biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like or even countervailing properties (e.g., antagonistic v. agonistic). It is thus contemplated that various changes may be made in the coding sequences of immunoglobulin-like domains without appreciable loss of the biological utility or activity of the encoded protein. It may even be possible to change particular residues in such domains to enhance their biological utility or to increase their interactive capability, for example, by increasing the binding affinity of Fc for RcRn.
As illustrated herein, transformed host cells will provide particularly good yields of immunoglobulin-like domains. The yields obtained are in the order of about 2 mg/L for CH3; 1-1.5 mg/L for CH2-hinge; 1.5-2 mg/L for Fc; and 0.5-1 mg/L for Fc-hinge. It is contemplated that such values may be readily scaled up to produce relatively large quantities of these domains in a matter of days, employing, for example, a (his)6 tag for affinity purification with Ni+2-NTA-agarose. Thus the expression system will provide a ready supply of immunoglobulin-like domain proteins which may be obtained in a relatively cost-effective manner.
Purification of immunoglobulin-like domains, such as native antibody constant domains, or Fc-hinge domains with increased half lives, may be achieved in many ways, including chromatography, density gradient centrifugation and electrophoretic methods.
The present invention facilitates the large scale production of immunoglobulin-like domains, including those derived from human sources, which may be employed in a wide variety of embodiments. These include their use in in vitro mutagenesis studies and in high resolution structural analyses, such as NMR and X-ray crystallography. Fc-hinge and Fc domain analyses have allowed the region involved in antibody catabolism to be delineated, showing that residues isoleucine (ile) 253, histidine (his) 310, his 435 and his 436 are important. Recombinant fragments, domains, or even subfragments thereof, may be used for mapping the Fc residues which are functionally important in binding to FcRn. Residues of recombinant Fc fragments may be altered, prior to expression as soluble proteins as disclosed herein, or on the surface of bacteriophage (McCafferty et al., 1990), and mutants binding with higher affinity to FcRn may be screened, or selected for, using solid surfaces coated with FcRn or FcRn in solution. The preferred method is to use FcRn in solution and then to capture FcRn:bacteriophage complexes on beads.
The large scale production of immunoglobulin Fc-hinge or Fc domains linked to other proteins or drugs also has potential for immunotherapy. In certain embodiments, chimaeric proteins or drugs may be produced which have the advantage of prolonged half lives and, since aglycosylated Fc has very low binding affinity for Fc receptors, they would not bind to the large number of immune cells that bear these receptors. This is a significant advantage since it reduces non-specific binding. Such aglycosylated Fc fragments will also not fix complement and, importantly, this would likely reduce the occurrence of local inflammatory reactions.
The present invention may also be described as a method of regulating IgG levels in serum comprising increasing FcRn binding to said IgG. This regulation may be accomplished by increasing or decreasing endogenous FcRn levels through alteration of the expression of FcRn, or by the use of recombinant cells expressing FcRn. In addition, the regulation may be accomplished by providing an FcRn with an altered binding affinity for IgG and thereby regulating IgG levels.
In a further embodiment the present invention may be extended to include other proteins, peptides or ligands, including non-protein ligands, that bind to FcRn with high affinity and in a pH dependent manner similar to that of the exemplary antibodies disclosed herein such that their serum half life is extended.
The present invention is exemplified by the production of large quantities of both variable region and constant region immunoglobulin-like domains, and genetically engineered mutant domains. Also included are examples of the production of immunoglobulin-like domains derived originally from an antibody molecule. In particular, the production of antibody Fc-hinge, Fc, CH2-hinge and CH3 domains; and Fc-hinge mutant domains with increased serum half lives, is disclosed. However, in light of such wide-ranging examples, which cover the spectrum of the immunoglobulin-like superfamily and modifications thereof, it will be understood that the present invention is not limited to these examples alone. Rather, it encompasses all the immunoglobulin-like structures described herein above.
In light of the previous discussion, the present invention may be described in certain broad aspects as a composition comprising a mutant IgG molecule having an increased serum half-life relative to IgG, and wherein said mutant IgG molecule has at least one amino acid substitution in the Fc-hinge region. The IgG may be any IgG molecule and is in certain embodiments, preferably a human IgG.
The invention may be also described in certain embodiments as a composition comprising a mutant IgG Fc-hinge fragment having an increased serum half-life relative to the serum half-life of IgG, and wherein said fragment has an increased binding affinity for FcRn. The compositions of the invention may thus comprise a molecule or fragment that has an amino acid substitution at one or more, or even three of the amino acids selected from number 252, 254, 256, 309, 311 or 315 in the CH2 domain or 433 or 434 in the CH3 domain, and in certain embodiments may have the following amino acid substitutions: leucine for threonine at position 252, serine for threonine at position 254 and phenylalanine for threonine at position 256. In the case of an antibody or particularly an IgG, increased binding affinity for FcRn may be defined as having a dissociation constant for binding to FcRn at pH 6, of less than about 7 nM as measured by surface plasmon resonance analysis. It is understood that any of the compositions of the present invention may also be defined in certain embodiments as pharmaceutically acceptable compositions.
In certain broad aspects, the invention may be described as a method of increasing the serum half-life of an agent comprising conjugating said agent to a mutant IgG or IgG Fc hinge fragment having an increased serum half life as described above. Preferred agents include, but are not limited to a therapeutic drug, an antigen binding polypeptide, an antigen or a receptor binding ligand, or even a T-cell receptor binding ligand, or a T-cell receptor domain.
The invention also encompasses a method of making an antibody with an increased serum half life comprising identifying a first amino acid in an IgG hinge region that is suspected of being directly involved in FcRn binding, identifying one or more second amino acids wherein each of said second amino acids is in the spatial region of said first amino acid, and wherein the side chain of said second amino acid is exposed to solvent in the native antibody, making an antibody with a random amino acid substitution of one or more of said second amino acids to make a mutant antibody, and identifying a mutant antibody having an increased serum half life. This method may further comprise the step of isolating the antibody. In the practice of the method, the first amino acid may be amino acid number 253, 310, 435 or 436 of the Fc fragment, and the second or secondary amino acid may be amino acid number 252, 254, 256, 309, 311 or 315 in the CH2 domain or 433 or 434 in the CH3 domain.
In certain broad aspects, the invention may be described as a composition comprising an Fc fragment comprising the fragment from about amino acid 250 to about amino acid 440 of an IgG antibody, further defined as having a higher binding affinity for FcRn than said IgG antibody, having one or more amino acid substitutions in a region near one or more FcRn binding amino acid residues and having a higher binding affinity for FcRn at pH 6 than at pH 7.4.
Another aspect of the present invention is a method of decreasing endogeneous serum IgG in a subject comprising administering to said subject an effective amount of the composition comprising proteins or peptides having increased serum half lives, and in particular administering an IgG with an increased serum half-life.
Certain embodiments of the invention also include methods of screening an agent for an increased serum half-life relative to the serum half-life of IgG, comprising the steps of obtaining a candidate agent, measuring the binding affinity of said agent to FcRn at pH 7.4 and at about pH 6, selecting a candidate agent with a higher binding affinity for FcRn at about pH 6 than at pH 7.4 and comparing the binding affinity of said selected agent to FcRn to the binding affinity of IgG to FcRn under identical conditions, wherein an increased binding affinity for FcRn relative to the binding affinity of IgG is indicative of an agent with an increased serum half-life. Certain preferred candidate agents may be a peptide or polypeptide, or even an antibody or a fragment of an antibody. In alternate embodiments the peptide may be selected from a random peptide library, or may be a randomly mutated protein, or even a synthetic peptide.
In certain embodiments, the invention may also be a method of increasing the serum half-life of a therapeutic agent comprising conjugating said therapeutic agent to an agent having an increased serum half-life relative to the serum half-life of IgG identified by the methods disclosed and claimed herein.